Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/19—Cytokines; Lymphokines; Interferons
  • A61K38/193—Colony stimulating factors [CSF]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • A61K38/1741—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals alpha-Glycoproteins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
  • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
  • A61K39/39541—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against normal tissues, cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
  • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/32—Cardiovascular disorders
  • G01N2800/329—Diseases of the aorta or its branches, e.g. aneurysms, aortic dissection
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the invention relates to aortopathy, and in particular, to compositions and methods for the diagnosis and treatment of aortopathy.
• Aortopathy relates to diseases of the aorta.
• Aortic dissection and intramural hematoma comprise a potentially life-threatening aortopathy involving separation of the aortic wall (1-6).
• the two conditions are distinguished by a tear in the aortic intima as present in the former classical form of aortic dissection which results in blood flow into the aortic wall and is absent in the latter form of intramural hematoma with bleeding confined within the aortic wall.
• This aortopathy is presently understood to be a continuum with the latter a variant and
• underpinnings e.g. ACTA2 (10,11)
• clinical/epidemiological aspects e.g. IRAD
• biochemical approaches e.g. smooth muscle biomarkers
• TGF and its downstream intracellular kinase signaling pathways play a
• KLF6 Kriippel-like factor 6
• GM-CSF granulocyte macrophage colony-stimulating factor
• GM-CSF is a neutralizing antibody against GM-CSF
• GM-CSF also acts as a diagnostic biomarker.
• GM-CSF granulocyte macrophage colony-stimulating factor
• a method of treating, preventing or ameliorating aortopathy in a subject comprising administering, to a subject in need of such treatment, a therapeutically effective amount of a granulocyte macrophage colony-stimulating factor (GM-CSF) negative modulator.
• GM-CSF granulocyte macrophage colony-stimulating factor
• the GM-CSF negative modulator is for treating, preventing or ameliorating an aortopathic condition selected from a group consisting of: aortic dissection; aortic intramural hematoma progression or recurrence; aortic aneurysm expansion, inflammation and/or rupture; and aortitis (i.e. aortic inflammation).
• the GM-CSF negative modulator may be used in maintenance treatment to prevent recurrent aortic dissection in subjects who have undergone primary surgical revision for acute aortic dissection.
• the GM-CSF negative modulator may be used in maintenance treatment to prevent aortic dissection progression in subjects with chronic aortic dissection, i.e.
• Granulocyte macrophage colony-stimulating factor (GM-CSF) or colony stimulating factor 2 (CSF2) is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells and fibroblasts and functions as a cytokine. It is a white blood cell growth factor, and GM-CSF signals via the signal transducer and activator of transcription, STAT5 and STAT3.
• GM-CSF human GM-CSF
• NC_ooooo5.io is provided herein as SEQ ID No:i, as follows:-
• SEQ ID No:2 One embodiment of the protein sequence (GenBank No: AAA52578.1; 144 amino acids) of human GM-CSF, is provided herein as SEQ ID No:2, as follows:-
• GM-CSF levels showed the greatest increase in macrophages derived from bone marrow of KLF6fl/fl;LysM Cre mice in response to Angll stimulation, as compared to control macrophages.
• Macrophages obtained from aorta of KLF6fl/fl;LysM Cre mice showed markedly increased expression of GM-CSF under experimental conditions of CaCl2 application and Angll infusion, and in macrophages derived from bone marrow of these mice.
• GM-CSF is therefore a neutralizing antibody which abrogated aortic dissection/intramural hematoma (Fig. 4a, b), as well as expression of GM-CSF receptor a, MMP9, F4/80 and IL-6 in addition to serum levels of IL-6.
• GM-CSF is therefore a neutralizing antibody which abrogated aortic dissection/intramural hematoma (Fig. 4a, b), as well as expression of GM-CSF receptor a, MMP9, F4/80 and IL-6 in addition to serum levels of IL-6.
• the GM-CSF negative modulator may be configured to:-
• the receptor through which of GM-CSF signalling is also known as Cluster of Differentiation 116, i.e. CD116. It is a heterodimer composed of an alpha and a beta chain. The alpha subunit contains a binding site for GM-CSF, and the beta chain is involved in signal transduction. Association of the two subunits results in receptor activation.
• the negative modulator comprises an anti-GM-CSF antibody or antigen-binding fragment thereof.
• anti-GM-CSF antibodies are well- known to the skilled person, and commercially available, for example from R&D Systems or Santa Cruz Biotechnology, Inc..
• the anti-GM-CSF antibody (as used in the Examples) is a mouse GM-CSF antibody (monoclonal rat IgG2A Clone #MPi22E9; catalogue number: MAB415 available from R&D Systems).
• the anti-GM-CSF antibody is catalogue number: sc-377039 (from Santa Cruz Biotechnology, Inc.).
• the anti-GM-CSF antibody or antigen-binding fragment thereof specifically binds to SEQ ID No: 2, or a variant or fragment thereof.
• GM-CSF antibody The epitope recognized by GM-CSF antibody from R&D Systems is unknown.
• the GM- CSF antibody from Santa Cruz (catalogue number: sc-377039) binds to an epitope mapping between amino acids 115-144 (TQIITFESFKENLKDFLLVIPFDCWEPVQE - SEQ ID No:3) at the C-terminus of GM-CSF of human origin.
• the anti-GM-CSF antibody or antigen-binding fragment thereof specifically binds to SEQ ID No:3, or a variant or fragment thereof.
• the antibody or antigen-binding fragment thereof may be monovalent, divalent or polyvalent. Preferably, the antibody or antigen-binding fragment thereof is isolated or purified.
• the antibody or antigen-binding fragment thereof comprises a polyclonal antibody, or an antigen-binding fragment thereof.
• the antibody or antigen-binding fragment thereof maybe generated in a rabbit, mouse or rat.
• the antibody or antigen-binding fragment thereof comprises a monoclonal antibody or an antigen-binding fragment thereof.
• the antibody of the invention is a human or humanised antibody.
• human antibody can mean an antibody, such as a monoclonal antibody, which comprises substantially the same heavy and light chain CDR amino acid sequences as found in a particular human antibody exhibiting immuno specificity for SEQ ID No:2, or a variant or fragment thereof.
• An amino acid sequence which is substantially the same as a heavy or light chain CDR, exhibits a considerable amount of sequence identity when compared to a reference sequence. Such identity is definitively known or recognizable as representing the amino acid sequence of the particular human antibody.
• Substantially the same heavy and light chain CDR amino acid sequence can have, for example, minor modifications or conservative substitutions of amino acids.
• Such a human antibody maintains its function of selectively binding to SEQ ID No:2 or a variant or fragment thereof.
• human monoclonal antibody can include a monoclonal antibody with substantially or entirely human CDR amino acid sequences produced, for example by recombinant methods such as production by a phage library, by lymphocytes or by hybridoma cells.
• humanised antibody can mean an antibody from a non-human species (e.g. mouse or rabbit) whose protein sequences have been modified to increase their similarity to antibodies produced naturally in humans.
• the antibody may be a recombinant antibody, i.e. a human antibody produced using recombinant DNA technology.
• the term "antigen-binding region” can mean a region of the antibody having specific binding affinity for its target antigen, for example, the peptide of SEQ ID No: 2, or a variant or fragment thereof.
• the fragment is an epitope, preferably SEQ ID No:3 or a variant or fragment thereof.
• the binding region may be a hypervariable CDR or a functional portion thereof.
• the term "functional portion" of a CDR can mean a sequence within the CDR which shows specific affinity for the target antigen.
• the functional portion of a CDR may comprise a ligand which specifically binds to SEQ ID No:2 or a fragment thereof.
• negative modulators according to the invention may be used in a monotherapy (e.g. the use of an antibody or antigen binding fragment thereof alone), for treating, ameliorating or preventing aortopathy.
• agents according to the invention may be used as an adjunct to, or in combination with, known therapies for treating, ameliorating, or preventing aortopathy, such as antihypertensives (beta blockers) and analgesia.
• known therapies for treating, ameliorating, or preventing aortopathy such as antihypertensives (beta blockers) and analgesia.
• the agents according to the invention may be combined in compositions having a number of different forms depending, in particular, on the manner in which the composition is to be used.
• the composition may be in the form of a powder, tablet, capsule, liquid, ointment, cream, gel, hydrogel, aerosol, spray, micellar solution, transdermal patch, liposome suspension or any other suitable form that may be administered to a person or animal in need of treatment.
• the vehicle of medicaments according to the invention should be one which is well-tolerated by the subject to whom it is given, and preferably enables delivery of the agents to the heart.
• Medicaments comprising agents of the invention may be used in a number of ways. For instance, oral administration may be required, in which case the agents may be contained within a composition that may, for example, be ingested orally in the form of a tablet, capsule or liquid. Compositions comprising agents and medicaments of the invention may be administered by inhalation (e.g. intranasally). Compositions may also be formulated for topical use. For instance, creams or ointments may be applied to the skin, for example adjacent to the heart.
• Agents and medicaments according to the invention may also be incorporated within a slow- or delayed-release device. Such devices may, for example, be inserted on or under the skin, and the medicament may be released over weeks or even months.
• the device may be located at least adjacent the treatment site, i.e. the heart. Such devices may be particularly advantageous when long-term treatment with agents used according to the invention is required and which would normally require frequent administration (e.g. at least daily injection).
• agents and medicaments according to the invention may be administered to a subject by injection into the blood stream or directly into a site requiring treatment.
• the medicament may be injected at least adjacent the heart. Injections may be intravenous (bolus or infusion) or subcutaneous (bolus or infusion), or intradermal (bolus or infusion).
• the amount of the agent e.g. anti-GM-CSF antibody
• the amount of the agent is determined by its biological activity and bioavailability, which in turn depends on the mode of administration, the physiochemical properties of the agent, and whether it is being used as a monotherapy or in a combined therapy.
• the frequency of administration will also be influenced by the half-life of the agent within the subject being treated.
• Optimal dosages to be administered may be determined by those skilled in the art, and will vary with the particular agent in use, the strength of the pharmaceutical composition, the mode of administration, and the advancement of the aortopathy. Additional factors depending on the particular subject being treated will result in a need to adjust dosages, including subject age, weight, gender, diet, and time of administration.
• a daily dose of between o.oo ⁇ g/kg of body weight and lomg/kg of body weight of agent according to the invention may be used for treating, ameliorating, or preventing aortopathy, depending upon which agent is used. More preferably, the daily dose of agent is between o.o ⁇ g/kg of body weight and lmg/kg of body weight, more preferably between o.nig/kg and and body weight.
• the agent may be administered before, during or after onset of aortopathy.
• Daily doses may be given as a single administration (e.g. a single daily injection).
• the agent may require administration twice or more times during a day.
• agents may be administered as two (or more depending upon the severity of the aortopathy being treated) daily doses of between 0.07 ⁇ g and 700 mg (i.e. assuming a body weight of 70 kg).
• a patient receiving treatment may take a first dose upon waking and then a second dose in the evening (if on a two dose regime) or at 3- or 4-hourly intervals thereafter.
• a slow release device may be used to provide optimal doses of agents according to the invention to a patient without the need to administer repeated doses.
• mice The dosage used for mice was 300 micrograms/mouse or 12 mg/kg every other day for 2 weeks by intra-peritoneal injection. Based on successful studies done in humans with intravenous MOR103 (1.0 or 1.5 mg/kg) once a week for 4 weeks and with subcutaneous mdressimumab (100 mg) every other week for 12 weeks, intravenous or subcutaneous administration of 75mg-ioomg once a week or every other week is preferred.
• Known procedures such as those conventionally employed by the pharmaceutical industry (e.g. in vivo experimentation, clinical trials, etc.), may be used to form specific formulations of the agents according to the invention and precise therapeutic regimes (such as daily doses of the agents and the frequency of administration).
• composition comprising a granulocyte macrophage colony-stimulating factor (GM-CSF) negative modulator; and optionally a pharmaceutically acceptable vehicle.
• GM-CSF granulocyte macrophage colony-stimulating factor
• the invention also provides in a fourth aspect, a process for making the pharmaceutical composition according to the third aspect, the process comprising combining a therapeutically effective amount of a GM-CSF negative modulator with a pharmaceutically acceptable vehicle.
• the negative modulator is preferably an antibody or antigen-binding fragment thereof, preferably an anti-GM-CSF antibody.
• a "subject” may be a vertebrate, mammal, or domestic animal.
• medicaments according to the invention may be used to treat any mammal, for example livestock (e.g. a horse), pets, or may be used in other veterinary applications.
• livestock e.g. a horse
• pets e.g. a human
• the subject is a human being.
• a “therapeutically effective amount” of the negative modulator is any amount which, when administered to a subject, is the amount of agent that is needed to treat the aortopathy disease, or produce the desired effect.
• the therapeutically effective amount of negative modulator used maybe from about 0.001 ng to about 1 mg, and preferably from about 0.01 ng to about 100 ng. It is preferred that the amount of negative modulator is an amount from about o.i ng to about 10 ng, and most preferably from about 0.5 ng to about 5 ng.
• a "pharmaceutically acceptable vehicle” as referred to herein, is any known compound or combination of known compounds that are known to those skilled in the art to be useful in formulating pharmaceutical compositions.
• the pharmaceutically acceptable vehicle may be a solid, and the composition may be in the form of a powder or tablet.
• a solid pharmaceutically acceptable vehicle may include one or more substances which may also act as flavouring agents, lubricants, solubilisers, suspending agents, dyes, fillers, glidants, compression aids, inert binders, sweeteners, preservatives, dyes, coatings, or tablet-disintegrating agents.
• the vehicle may also be an encapsulating material.
• the vehicle is a finely divided solid that is in admixture with the finely divided active agents according to the invention.
• the active agent may be mixed with a vehicle having the necessary compression properties in suitable proportions and compacted in the shape and size desired.
• the powders and tablets preferably contain up to 99% of the active agents.
• suitable solid vehicles include, for example calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• the pharmaceutical vehicle may be a gel and the composition may be in the form of a cream or the like.
• the pharmaceutical vehicle may be a liquid, and the pharmaceutical composition is in the form of a solution.
• Liquid vehicles are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
• the active agent according to the invention may be dissolved or suspended in a pharmaceutically acceptable liquid vehicle such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid vehicle can contain other suitable pharmaceutical additives such as solubilisers, emulsifiers, buffers, preservatives, sweeteners, flavouring agents, suspending agents, thickening agents, colours, viscosity regulators, stabilizers or osmo-regulators.
• suitable examples of liquid vehicles for oral and parenteral administration include water (partially containing additives as above, e.g.
• cellulose derivatives preferably sodium carboxymethyl cellulose solution
• alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, and oils (e.g. fractionated coconut oil and arachis oil).
• the vehicle can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile liquid vehicles are useful in sterile liquid form compositions for parenteral administration.
• the liquid vehicle for pressurized compositions can be a halogenated hydrocarbon or other pharmaceutically acceptable propellant.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions, can be utilized by, for example, intramuscular, intrathecal, epidural, intraperitoneal, intravenous and particularly subcutaneous injection.
• the agent may be prepared as a sterile solid composition that may be dissolved or suspended at the time of administration using sterile water, saline, or other appropriate sterile injectable medium.
• compositions of the invention may be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (for example, enough saline or glucose to make the solution isotonic), bile salts, acacia, gelatin, sorbitan monoleate, polysorbate 80 (oleate esters of sorbitol and its anhydrides copolymerized with ethylene oxide) and the like.
• the agents used according to the invention can also be administered orally either in liquid or solid composition form.
• Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders, and liquid forms, such as solutions, syrups, elixirs, and suspensions.
• Forms useful for parenteral administration include sterile solutions, emulsions, and suspensions.
• the GM-CSF is elevated in subject's suffering from aortopathy conditions.
• GM-CSF granulocyte macrophage colony-stimulating factor
• SEQ ID No:2 acts as a suitable biomarker, which may be detected.
• SEQ ID No: 2 acts as an epitope which may be bound by an antibody or antigen-binding fragment, preferably the antibody or antigen-binding fragment used in accordance with the first aspect.
• the invention also provides a kit for diagnosing patients suffering from aortopathy.
• kits for diagnosing a subject suffering from aortopathy, or a pre-disposition thereto, or for providing a prognosis of the subject's condition comprising:
• detection means for detecting, in a sample obtained from a test subject, the concentration of granulocyte macrophage colony-stimulating factor (GM-CSF), or a variant or fragment thereof, and
• kit is configured to identify a difference in the concentration of GM-CSF in the bodily sample from the test subject compared to the reference, thereby suggesting that the test subject is suffering from aortopathy, or has a pre-disposition thereto, or providing a negative prognosis of the subject's condition.
• concentration of GM-CSF in an individual who does not suffer from aortopathy wherein a difference in the concentration of GM-CSF in the bodily sample from the test subject compared to the reference suggests that the subject is suffering from aortopathy, or has a pre-disposition thereto, or provides a negative prognosis of the subject's condition.
• the subject may be any animal of veterinary interest, for instance, a cat, dog, horse etc.
• the subject is a mammal, such as a human, either male or female.
• a sample is taken from the subject, and the concentration of granulocyte macrophage colony-stimulating factor (GM-CSF), or a variant or fragment thereof maybe measured in an assay.
• the kit may comprise sample extraction means for obtaining the sample from the test subject.
• the sample extraction means may comprise a needle or syringe or the like. It has been demonstrated that GM-CSF occur in body and organ fluids.
• the sample may be any bodily sample into which GM-CSF is secreted, e.g. it may be lymph or interstitial fluid.
• the sample may be a urine sample or a blood sample.
• the blood sample may be venous or arterial.
• the kit may comprise a sample collection container for receiving the extracted sample.
• "fresh" bodily samples such as blood
• the blood may be stored at low temperatures, for example in a fridge or even frozen before the GM-CSF assay is conducted. The sample may then be de-frosted and analysed at a later date.
• Measurement of GM-CSF may be made on whole blood.
• the blood may be further processed before the assay is performed.
• an anticoagulant such as citrate (such as sodium citrate), hirudin, heparin, PPACK, or sodium fluoride may be added.
• the sample collection container may contain an anticoagulant in order to prevent the blood sample from clotting.
• the blood sample may be centrifuged or filtered to prepare a plasma or serum fraction, which may be used for analysis.
• the GM-CSF or variant or fragment is analysed or assayed in a blood plasma or a blood serum sample. It is preferred that GM-CSF concentration is measured in vitro from a blood serum sample or a plasma sample taken from the subject.
• the inventors monitored the concentration of GM-CSF in
• KLF6fl/fl;LysM Cre mice under Angll infusion to induce aortic inflammation and compared it to the GM-CSF concentration in control macrophages. They demonstrated that there was a statistically significant increase in the concentration of GM-CSF in the mice suffering from aortopathy induced by Angll infusion. Thus, the difference in concentration is preferably an increase compared to the reference control value, which is indicative of aortopathy.
• the concentration of GM-CSF in aortopathy patients is highly dependent on a number of factors, for example how far the disease has progressed, and the age and gender of the subject. It will also be appreciated that the concentration of GM-CSF in individuals who do not suffer from aortopathy may fluctuate to some degree, but that on average over a given period of time, the concentration tends to be substantially constant. In addition, it should be appreciated that the concentration of GM-CSF in one group of individuals who do not suffer from aortopathy may be different to the concentration of GM-CSF in another group of individuals who do not suffer from the disease.
• the skilled technician will know how to determine the average concentration of GM-CSF in individuals who do not suffer from aotopathy, and this is referred to as the 'normal' concentration of GM-CSF.
• the normal concentration corresponds to the reference values discussed above.
• the GM-CSF may be extracted from the bodily sample by a variety of techniques, and then detected. Detection may be achieved by an immunoassay (e.g. ELISA), for example using a monoclonal antibody specific for GM-CSF which has been pre-coated onto a substrate, such as a microplate. Standards and samples maybe applied into the wells and any GM-CSF present is bound by the immobilised antibody. After washing away any unbound substances, an enzyme- linked monoclonal antibody for human GM-CSF may then be applied to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution may be applied to the wells and colour develops in proportion to the amount of GM-CSF bound in the initial step. The colour development may then be stopped, and the intensity of colour measured.
• a suitable assay for detecting GM-CSF may be as supplied by R&D systems.
• the kit according to the invention comprises means for determining the concentration of GM- CSF in the bodily sample.
• the kit may comprise a container in which the means for determining the concentration of GM-CSF in the sample from a test subject may be contained.
• the kit may also comprise instructions for use.
• the kit may comprise detection means for determining the concentration of the GM-CSF in the sample once this has been obtained from the subject.
• the reference values may be obtained by assaying a statistically significant number of control samples (i.e. samples from subjects who do not suffer from aortopathy). Accordingly, the reference according to the kit of the invention may be a control sample for assaying purposes.
• the detection means may comprise an assay adapted to determine the concentration of GM-CSF in the sample.
• the kit or method may comprise the use of a positive control and/or a negative control against which the assay may be compared.
• the kit may comprise a reference for the concentration of GM-CSF in a sample from an individual who does (i.e.
• the kit may further comprise a label which may be detected.
• label can mean a moiety that can be attached to the antibody, or antigen binding fragment thereof. Moieties can be used, for example, for therapeutic or diagnostic procedures. Labels include, for example, moieties that can be attached to an anti-GM-CSF antibody or fragment thereof and used to monitor the binding of the antibody to GM-CSF or a fragment thereof. As described herein the antibody or antigen-binding fragment thereof binds specifically to SEQ ID No:2, or a variant or fragment thereof.
• Diagnostic labels include, for example, moieties which can be detected by analytical methods.
• Analytical methods include, for example, qualitative and quantitative procedures.
• Qualitative analytical methods include, for example, immunohistochemistry and indirect
• Quantitative analytical methods include, for example, immuno affinity procedures such as radioimmunoassay, ELISA or FACS analysis. Analytical methods also include both in vitro and in vivo imaging procedures. Specific examples of diagnostic labels that can be detected by analytical means include enzymes, radioisotopes, fluorochromes, chemiluminescent markers, and biotin.
• a label can be attached directly to an anti-GM-CSF antibody, or antigen binding fragment thereof, or be attached to a secondary binding agent that specifically binds a molecule of the invention.
• a secondary binding agent can be, for example, a secondary antibody.
• a secondary antibody can be either polyclonal or monoclonal, and of human, rodent or chimeric origin.
• an anti-GM-CSF antibody for use in the treatment of a patient suffering from aortic dissection, wherein said antibody is administered to said patient in a manner to achieve a therapeutically effective antibody level in the blood of said patient.
• a method of treating aortic dissection in a patient by measuring the presence of an increased level of GM-CSF in the blood compared to normal and then treating said patient with an anti-GM-CSF antibody.
• the patient may have been diagnosed with aortic dissection and requires ongoing treatment to prevent further progression of the disease.
• the patient may have undergone surgical treatment for aortic dissection and requires ongoing treatment to prevent recurrence of the disease.
• a method of diagnosing patients suffering from or at risk of suffering from aortic dissection comprising analysing the concentration of GM-CSF in a patient sample and comparing this concentration with a reference concentration of GM-CSF known to represent an elevated risk of aortic dissection.
• the patient may have been diagnosed with aortic dissection and requires ongoing treatment to prevent further progression of the disease.
• the patient may have undergone surgical treatment for aortic dissection and requires ongoing treatment to prevent recurrence of the disease.
• the patient sample may comprise a blood sample.
• nucleic acid or peptide or variant, derivative or analogue thereof which comprises or consists of substantially the amino acid or nucleic acid sequences of any of the sequences referred to herein, including variants or fragments thereof.
• substantially the amino acid/nucleotide/peptide sequence can be a sequence that has at least 40% sequence identity with the amino acid/nucleotide/peptide sequences of any one of the sequences referred to herein, for example 40% identity with the sequence identified as SEQ ID No: 2 (i.e. GM-CSF protein), and so on.
• amino acid/polynucleotide/polypeptide sequences with a sequence identity which is greater than 50%, more preferably greater than 65%, 70%, 75%, and still more preferably greater than 80% sequence identity to any of the sequences referred to are also envisaged.
• the amino acid/polynucleotide/polypeptide sequence has at least 85% identity with any of the sequences referred to, more preferably at least 90%, 92%, 95%, 97%, 98%, and most preferably at least 99% identity with any of the sequences referred to herein.
• the skilled technician will appreciate how to calculate the percentage identity between two amino acid/polynucleotide/polypeptide sequences.
• the percentage identity for two sequences may take different values depending on:- (i) the method used to align the sequences, for example, ClustalW, BLAST, FASTA, Smith-Waterman
• the alignment method for example, local vs global alignment, the pair-score matrix used (e.g. blosum62, pam250, gonnet etc.), and gap-penalty, e.g. functional form and constants.
• the pair-score matrix used e.g. blosum62, pam250, gonnet etc.
• gap-penalty e.g. functional form and constants.
• percentage identity between the two sequences. For example, one may divide the number of identities by: (i) the length of shortest sequence; (ii) the length of alignment; (iii) the mean length of sequence; (iv) the number of non-gap positions; or (iv) the number of equivalenced positions excluding overhangs. Furthermore, it will be appreciated that percentage identity is also strongly length dependent. Therefore, the shorter a pair of sequences is, the higher the sequence identity one may expect to occur by chance.
• a substantially similar nucleotide sequence will be encoded by a sequence which hybridizes to any of the nucleic acid sequences shown herein, or their complements under stringent conditions.
• stringent conditions we mean the nucleotide hybridises to filter-bound DNA or RNA in 3x sodium chloride/sodium citrate (SSC) at approximately 45°c followed by at least one wash in o.2x ssc/o.i SDS at approximately 20-65°c.
• a substantially similar polypeptide may differ by at least 1, but less than 5, 10, 20, 50 or 100 amino acids from the sequences shown herein.
• nucleic acid sequence described herein could be varied or changed without substantially affecting the sequence of the protein encoded thereby, to provide a functional variant thereof.
• Suitable nucleotide variants are those having a sequence altered by the substitution of different codons that encode the same amino acid within the sequence, thus producing a silent change.
• Other suitable variants are those having homologous nucleotide sequences but comprising all, or portions of, sequence, which are altered by the substitution of different codons that encode an amino acid with a side chain of similar biophysical properties to the amino acid it substitutes, to produce a conservative change.
• small non-polar, hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine.
• Large non-polar, hydrophobic amino acids include phenylalanine, tryptophan and tyrosine.
• the polar neutral amino acids include serine, threonine, cysteine, asparagine and glutamine.
• the positively charged (basic) amino acids include lysine, arginine and histidine.
• the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. It will therefore be appreciated which amino acids may be replaced with an amino acid having similar biophysical properties, and the skilled technician will know the nucleotide sequences encoding these amino acids.
• Figure l shows aortic aneurysm and inflammation in KLF6 heterozygous knockout mice,
• aorta infrarenal aorta:hash, suprarenal aorta: asterisk
• WT wild-type
• Figure 2 shows myeloid deficiency of KLF6 shows a phenotype of aortic
• Figure 3 shows GM-CSF is a direct target of KLF6 in macrophages, (a) RT2 profiler
• BM bone marrow
• n 6]. N.D. indicates not detected, (c) mRNA expression of GM-CSF in aorta of
• Figure 4 shows GM-CSF is required for aortic dissection/intramural hematoma
• Figure 5 shows increased GM-CSF in patients with acute aortic dissection,
• CAD coronary artery disease
• b Immunofluorescent staining for CD68 (red, c), GM-CSF (green, d) and DAPI (blue, e) in descending dissected aorta (boxed area, a) with EVG staining (b);
• Figure 6 shows aortic dissection/intramural hematoma on aneurysm in the present model, (a) Appearance of the excised thoracic- abdominal aorta subjected to CaCl2 application and Angll infusion. Note that intramural thrombus formation is present in the suprarenal region, (b) Schematic illustration of the diseased aorta, (c) Cross-sectional histological sections stained by Elastica van Gieson.
• a-e in correspond to the same levels of the aorta (a), a; Cross section of the infrarenal abdominal aorta (CaCl2 application level), b; at the level of the renal arteries, c; suprarenal level where the intima-medial layer shows a tear, d and e;
• Figure 7 shows marked infiltration of macrophages in the aneurysmal aorta
• Infiltrated macrophages were visualized by immunofluorescent staining (dotted line, green, Mac3) in aorta of KLF6fl/fl;LysMCre mice (right panels, c and d) compared to KLF6fl/fl mice (left panels, a and b).
• Figure 9 shows effects of LysM Cre on neutrophils and dendritic cells in peripheral blood.
• Figure io shows GM-CSF is regulated by KLF6 in macrophages.
• Macrophages from KLF6fl/fl mice were infected with empty (E) or KLF6 (KLF6)-expressing retrovirus construct.
• Total RNA was harvested 3h after stimulation with Angll (10 mM), TNFa (10 ng ml-i) and IL- ⁇ (20 ng ml- 1).
• CTL control
• Angll 10 mM
• TNFa 10 ng ml-i
• IL- ⁇ 20 ng ml-i
• Figure 13 shows effects of LysM Cre on neutrophils and dendritic cells in the aorta.
• Ly6G+ neutrophils a, c
• CDiic+MHC+ dendritic cells DC
• b, d CDiic+MHC+ dendritic cells
• mice were anesthetized and underwent laparotomy at 10- to 13-weeks of age.
• the abdominal aorta between the renal arteries and bifurcation of the iliac arteries was isolated from the surrounding retroperitoneal structure, and 0.5 M CaCl2 was applied to the external surface of the infrarenal aorta. NaCl (0.9%) was substituted for CaCl2 in sham control mice. The aorta was rinsed with 0.9% sterile saline after 15 min and the incision was closed.
• Wild-type mice were injected intraperitoneally with 110 mg kg-i of clodronate liposomes or equal volume of PBS liposomes 2 days prior and 7 days after induction of aortic dissection.
• Neutralizing antibody against GM-CSF (300 ⁇ g, R&D systems) or control anti-rat IgG antibody (Equitech Bio) was administered every other day by intraperitoneal injection.
• Recombinant murine GM-CSF (10, 50, 100 ⁇ g kg-i day-i, PeproTech) was administered for two weeks or four weeks after induction of aortic dissection. Histological analysis and immunohistochemistry
• Aortas from mice were embedded in paraffin then 5 ⁇ m-thick serial sections were prepared for Elastic Von Gienson (EVG) and hematoxylin/eosin (HE) staining. Digital images of EVG-stained aortas with reference scale were used for absolute measurement of diameter. Human aortic tissue was obtained from patients undergoing surgical aortic repair with informed consent under a protocol approved by the University of Tokyo hospital research ethics committee. Paraffin-embedded sections were taken from the aorta for EVG staining
• Aortas were minced into 3- to 4-mm pieces and placed in 1 ml digestion solution containing collagenase type II (1.25 mg ml-i, Worthington) and porcine pancreatic elastase (50 ⁇ g ml-i, Worthington) in base solution of Accumax (Innovative Cell Technologies).
• Aortic tissue was digested at room temperature with agitation for 1 h. After digestion, cells were washed in FACS buffer (5% FCS in PBS) at 2000 rpm for 5 min. Aortic
• Bone marrow-derived cells were taken from the femur and tibia of 5- to 6-week-old mice. Blood was collected in heparin-coated
• Neutrophils were isolated from bone marrow using a neurophil isolation kit according to the manufacturer's instructions (Miltenyi Biotec). From single-cell suspensions of spleen, bone marrow and blood, erythrocytes were lysed using ACK lysis buffer for 5, 3 and 2 min on ice, respectively. Cells were centrifuged at 2000 rpm for 5 min to remove the ACK lysis buffer, then the single-cell suspensions were resuspended and washed in FACS buffer, followed by centrifugation at 2000 rpm for 5 min.
• Bone marrow-derived cells were prepared from femur and tibia of KLF6fl/fl mice or
• KLF6fl/fl;LysM Cre mice to assess the role of GM-CSF in macrophages.
• KLF6 overexpression was induced by retrovirus construct for KLF6 (pMXs-KLF6) in the presence of RetroNectin (5 ⁇ g/cm2, Takara Bio.).
• Murine Fc receptors were blocked using antibodies against murine CD16/32 antigens
• FITC-CD3e[i45-2Cn], FITC-Ly6G[RB6-8Cs], FITC- CDnb[Mi/70], FITC-CD45R/B220[RA3-6B2] and FITC-Ly76[Ter-ii9] were used as lineage markers.
• Corresponding isotype control antibodies were added to samples at the same concentrations as the antibodies of interest. After incubation,
• ChIP analysis was performed using a Chromatin Immunoprecipitation Kit (Active Motif) according to the manufacturer's instructions. Briefly, bone marrow-derived macrophages were stimulated with or without Angll (10 ⁇ ), TNFa (1 0 ng ml-i) and IL- ⁇ (20ng ml-i) for 3 h prior to crosslinking for 10 min with 1% formaldehyde. Chromatin was sheared by sonication to an average size of 200 ⁇ iooo base pairs (Covaris). Immunoprecipitation was performed using anti-KLF6 antibody (Santa Cruz Biotechnology) and rabbit IgG antibody (Santa Cruz Biotechnology).
• KLF-binding elements was performed using the following primers: forward: 5'-AAGC CCTTCCAAGAACTGGC-3' (SEQ ID NO: 4) and reverse 5'-GGCCCCTCAAAAAGGAGAGG-3' (SEQ ID NO: 5) ⁇
• KLF6 recruitment was normalized by input DNA and compared to control group with
• RNA from cultured cells, aortic macrophages, bone marrow-derived neutrophils or murine aortic samples was extracted using either RNeasy minikit (Qiagen) or RNAlater (Qiagen) according to the manufacturer's instructions.
• RNAlater Qiagen
• 0.5 ⁇ g-l ⁇ g RNA was reverse-transcribed using Superscript III (Invitrogen) according to the manufacturer's instructions.
• PCR reactions were performed using 2 ⁇ of resulting cDNA per 20 ⁇ reaction volume containing SYBR green I master (Roche). GAPDH was used as an internal control.
• RT2 Using bone marrow-derived macrophages with Angll (10 ⁇ , 3h) stimulation, RT2
• PCR was performed on a LightCycler 480 Real-time PCR
• Protein bands were detected by ECLplus (Thermo scientific) and GAPDH served as an internal control for protein loading.
• Plasma levels of IL-6, MCP-i and GM-CSF in mice or in humans with or without aortic dissection/intramural hematoma were assayed with commercially available quantikine ELISA kits (R&D systems) according to the manufacturer's instructions.
• Sera of healthy volunteers and of patients with aortic aneurysm, coronary artery disease or with aortic dissection were obtained with informed consent under a protocol approved by the University of Tokyo hospital research ethics committee. Baseline characteristics of human subjects are shown in Table 6.
• Example 1 Aortic aneurysm with inflammation in KLF6 heterozygous knockout mice
• mice heterozygously depleted for KLF6 manifest a phenotype of exacerbated aortic aneurysm (defined as greater than 50% increase in external aortic diameter with conserved aortic wall) (21,22) when subjected to aortic inflammation [two weeks-infusion of angiotensin II (Angll) with local application of calcium chloride
• MMP9 matrix metalloprotease-9
• F4/80 as a marker of macrophages
• IL-6 as a marker of inflammation
• Example 2 KLF6fl/fl:LysM Cre mice exhibit aortic dissection/hematoma
• mice were further generated which showed specific reduction of KLF6 expression in the myeloid lineage by 70% as compared to control mice.
• KLF6fl/fl;LysM Cre mice subjected to aortic inflammation showed a similar phenotype of exacerbated abdominal aortic aneurysm to that seen in heterozygous knockout mice, but intriguingly, further showed supra-renal aortic dissection/intramural hematoma as defined as separation of the intra- aortic wall with hematoma formation accompanied by intimal tear for dissections (Fig.6).
• This lesion also showed fibrotic tissue deposition with infiltration of Mac3-positive macrophages (Fig.
• KLF6fl/fl;LysM Cre mice showed elevated expression of IL-6 in
• Fig. 2h Immune cells in the diseased aorta of these mice were characterized by flow cytometry analysis which showed a markedly increased population of CDnb+Ly6Chi inflammatory monocytes and this increase was also seen in the peripheral blood (Fig. 21).
• Granulocytes e.g. neutrophils;Ly6G+ cells
• TGF a central molecule in the pathogenesis of Marfan aortopathy (12, 14, 32-34), and its downstream signaling pathways (canonical pSmad-235 and non-canonical
• Example 3 - GM-CSF is a downstream target of KLF6
• GM-CSF levels showed the greatest increase in macrophages derived from bone marrow of KLF6fl/fl;LysM Cre mice in response to Angll stimulation (3.89-fold) as compared to control macrophages (Fig. 3a).
• macrophages obtained from aorta of KLF6fl/fl;LysM Cre mice showed markedly increased expression of GM-CSF under experimental conditions of CaCl2 application and Angll infusion (Fig. 3b), and in macrophages derived from bone marrow of these mice (Fig. 10a). Expression of GM-CSF in the aorta was elevated from
• GM-CSF is a direct target of KLF6 and that KLF6 represses expression of GM-CSF.
• Example 4 GM-CSF manipulation regulates aortic dissection/hematoma
• the inventors further investigated whether GM-CSF is sufficient to induce the aortopathy.
• lymphocytes did not change in either the early phase (5 days) or developed phase (14 days) of the model (Tables 2 and 3). With respect to neutrophils, the
• circulating levels of GM-CSF were measured in sera of patients with acute aortic dissection which showed marked increases in contrast to patients with coronary artery disease, aortic aneurysm or healthy volunteers which
• GM-CSF is associated with aortic acute dissection not only in mice but also in human conditions.
• GM-CSF is a key regulatory molecule causative of
• mice modulation of GM-CSF by a
• GM-CSF was a central component of the aortic dissection/intramural hematoma
• TGF -SMAD pathway is sufficient to trigger this condition in a model of inflammation and degenerative aorta (calcium chloride treatment causes stiffening of the aorta to
• GM-CSF tissue expression had also been shown to be increased in a patient presenting with aortic dissection in Cogan's disease (41), an apparently auto-immune condition which is characterized by recurrent corneal inflammation (42) that was thought to be an isolated finding.
• dendritic cells CDiic+MHCII+ cells
• M-CSF Macrophage colony-stimulating factor
• GM-CSF are envisioned given different expression patterns in the vascular wall.
• M-CSF is constitutively expressed under physiological conditions in
• endothelial cells fibroblasts, macrophages and smooth muscle cells, GM-CSF, by
• TNF inflammatory stimuli
• LDL lipoprotein
• M-CSF is a constitutively expressed cytokine in the vasculature
• GM-CSF is markedly induced in diseased vessels to regulate pathological conditions including the described aortopathy.
• this model might involve hemodynamic stress on the supra-renal
• GM-CSF is a central regulator of aortic dissection/intramural hematoma in the atherosclerotic and inflammatory aorta which is typically seen in the elderly patient with this condition, and may serve as a potential target for diagnostic and therapeutic exploitation (e.g. aortic stabilization using
• GM-CSF antagonists as well as a diagnostic biomarker.
• Aortic dissection and intramural hematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear.
• GM-CSF granulocyte macrophage colony-stimulating factor
• KLF6 myeloid-specific conditional deficient mice exhibited this aortic phenotype when subjected to aortic inflammation.
• KLF6 down-regulated expression and secretion of GM-CSF.
• Administration of a neutralizing antibody against GM-CSF prevented the condition in these mice.
• GM-CSF in combination with aortic inflammation to wild-type mice was sufficient to induce the phenotype suggesting the general nature of effects. Moreover, patients with this condition showed highly increased circulating levels of GM-CSF, which was also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory

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